Increasing the data size to accurately reconstruct the phylogenetic relationships between nine subgroups of the Drosophila melanogaster species group (Drosophilidae, Diptera).

Previous phylogenetic analyses of the melanogaster species group have led to conflicting hypotheses concerning their relationship; therefore the addition of new sequence data is necessary to discover the phylogeny of this species group. Here we present new data derived from 17 genes and representing 48 species to reconstruct the phylogeny of the melanogaster group. A variety of statistical tests, as well as maximum likelihood mapping analysis, were performed to estimate data quality, suggesting that all genes had a high degree of contribution to resolve the phylogeny. Individual locus was analyzed using maximum likelihood (ML), and the concatenated dataset (12,988 bp) were analyzed using partitioned maximum likelihood (ML) and Bayesian analyses. Separated analysis produced various phylogenetic relationships, however, phylogenetic topologies from ML and Bayesian analysis based on concatenated dataset, at the subgroup level, were completely identical to each other with high levels of support. Our results recovered three major clades: the ananassae subgroup, followed by the montium subgroup, the melanogaster subgroup and the oriental subgroups form the third monophyletic clade, in which melanogaster (takahashii, suzukii) forms one subclade and ficusphila [eugracilis (elegans, rhopaloa)] forms another. However, more data are necessary to determine the phylogenetic position of Drosophila lucipennis which proved difficult to place.

Convergent intron loss of MRP1 in Drosophila and mosquito species.

Previous study revealed that the MRP1 gene ortholog DMRP1/CG6214 of Drosophila melanogaster contains 12 exons in the coding region. In the current study, the genes of DMRP1/CG6214 from D. melanogaster and Drosophila virilis were compared, and the result indicated that D. virilis had an extra intron located in exon 2, implying that intron loss or gain might have occurred at this locus. To track the evolution of the extra intron (Intron Z), orthologous nucleotide sequences of 37 arthropod species were cloned or annotated. Based on phylogenetic analysis, we found that Intron Z should present in the common ancestor of arthropod species, more than 420 Ma. In addition, we found that Sophophora subgenus species and mosquito (Culex pipiens) lost Intron Z independently, showing evolutionary convergence.

An intron loss of Dfak gene in species of the Drosophila melanogaster subgroup and phylogenetic analysis.

Drosophila focal adhesion kinase (Dfak) gene is a single-copy nuclear gene. Previous study revealed that Drosophila melanogaster and Drosophila simulans had lost an intron precisely within the tyrosine kinase (TyK) domain of this gene. However, this did not happen in several other Drosophila species, including Drosophila elegans, Drosophila ficusphila, Drosophila biarmipes, Drosophila jambulina, Drosophila prostipennis, Drosophila takahashii, and Drosophila pseudoobscura. In the current study, homologous sequences of Drosophila sechellia, Drosophila mauritiana, Drosophila yakuba, Drosophila teissieri, Drosophila santomea, and Drosophila erecta were amplified by polymerase chain reaction, and further sequencing analysis indicated that these species were missing a TyK domain intron, indicating they were closely related. The relationship of the D. melanogaster species group was reconstructed using TyK domain nucleotide sequences. The resulting phylogenetic tree revealed that these 8 species were the most related species in the melanogaster group. These results strongly support previously proposed classifications based on morphological and molecular data.

Research on the karyotype and evolution of Drosophila melanogaster species group.

Mitotic metaphase chromosomes of 34 species of Drosophila melanogaster species group were examined. Certain new karyotypes were described for the first time, and their evolutionary and interspecific genetic relationships among 8 subgroups of D. melanogaster species group were analyzed systematically. The results were as follows. The basic karyotype of elegans subgroup was type A. The karyotypes of eugracilis subgroup, melanogaster subgroup, and ficusphila subgroup were all type C. The karyotypes of takahashii subgroup and suzukii subgroup were both type C and type D. The montium subgroup had six kinds of karyotypes: types B, C, C', D, D', and E. The ananassae subgroup had three kinds of karyotypes: types F, G, and H. Thus, the melanogaster species group was classified into five pedigrees based on the diversity of these karyotypes: 1) elegans; 2) eugracilis-melanogaster-ficusphila; 3) takkahashii-suzukii; 4) montium; 5) ananassae. The above-mentioned results in karyotypic evolution were consistent with those of DNA sequence analysis reported by Yang except for the elegans subgroup and this subgroup was considered as the ancestral subgroup. Karyotype analysis of the same drosophila from different isofemale lines indicated that the same Drosophila from different places showed karyotypic variation which might be due to different geographical environment and evolutionary degree or interaction between the two factors.

[Karyotypes of five species in the Drosophila melanogaster species group].

Based on the traditional knocking-slides and Giemsa-staining methods,this paper firstly analyzed the metaphase chromosomes of five species (D. constricta, D. ohnishii, D. ogumai, D. pseudobaimaii and D. tani) of Drosophila melanogaster species group. The results show that all of the five species share the same chromosomal numbers (2n=8) but with different typical shapes. D. pseudobaimaii and D. tani share the shape 2V, 1R, 1D; D. constricta shows 2V,1R,1D with unclear recognized dot chromosomes; D. ohnishii and D. ogumai share the same chromosomal shapes 2V2R. And that between karyotype and phylogenetic relationships are found to be correspond.

[Three steps for amplifying quick evolving region of DNA.].

A common problem in research of molecular evolution is difficult to efficiently amplify quick evolving target sequence of genes in different species or genus using specific primers, thus making experimental process and final analysis of total results delayed. Although using nested or semi-nested PCR can prominently increase PCR specificity, it really cannot efficiently amplify quick evolving region of DNA in our research of gene Fak56D. In this research, we need PCR products corresponding to gene Fak56D of different species of Drosophila melanogaster or other genus of Drosophila . For the high evolutionary rate,most materials did not produce qualified PCR products. To solve this problem, we initially used a combination method of three steps, i.e. semi-nested PCR taken together with orientational gel extraction, which satisfactorily met the demands of next cloning and sequencing steps.

Phylogenetic relationships of Drosophila melanogaster species group deduced from spacer regions of histone gene H2A-H2B.

Nucleotide sequences of the spacer region of the histone gene H2A-H2B from 36 species of Drosophila melanogaster species group were determined. The phylogenetic trees were reconstructed with maximum parsimony, maximum likelihood, and Bayesian methods by using Drosophila pseudoobscura as the out group. Our results show that the melanogaster species group clustered in three main lineages: (1). montium subgroup; (2). ananassae subgroup; and (3). the seven oriental subgroups, among which the montium subgroup diverged first. In the third main lineage, suzukii and takahashii subgroups formed a clade, while eugracilis, melanogaster, elegans, ficusphila, and rhopaloa subgroups formed another clade. The bootstrap values at subgroup levels are high. The phylogenetic relationships of these species subgroups derived from our data are very different from those based on some other DNA data and morphology data.

[Random amplified polymorphic DNA (RAPD) analysis of Drosophila simulans in the mainland of China].

The widespread distribution of Drosophila simulans in the mainland of China was found recently. Random amplified polymorphic DNA (RAPD) was used to analyzed DNA polymorphisms of 38 Drosophila simulans populations collected from the mainland of China. The origins of D. simulans in China were also discussed. Using 40 arbitary primers (10 bp), we made PCR amplifications under the optimized reaction conditions for RAPD that had been established in our laboratory. Our molecular phylogenetic tree constructed by UPGMA method showed as follows: (1) 38 populations are apparently classified into Northern cluster and Southern cluster, where NJ(Nanjing) can be considered as the boundary according to latitude. The populations in the Northern cluster can be further grouped into two sub-clusters, where BJ(Beijing) can be considered as the boundary according to latitude. The Northeast one consists of 10 populations, including Mohe(MH), Hailaer(HLR), Heihe(HH), Jiamusi(JMS), Haerbin(HRB), Changchun(CC), Shenyang(SY), Dandong(DD), Yanji(YJ), Tumen(TM); the other consists of 12 populations, including Beijing (BJ), Datong(DT), Huhehaote(HHHT), Yinchun(YC), Xinin(XN), Lanzhou(LZ), Taiyuan(TY), Shijiazhuang(SJZ), Yantai (YT), Jinan(JN), Xuzhou(XZ), Lianyungang(LYG). The southern cluster consists of the left 16 populations, including Zhengzhou (ZZ), Wuhan(WH), Shanghai(SH), Nanjing(NJ), Hangzhou(HZ), Chongqing(CQ), Nanchang(NC), Wenzhou(WZ), Changsha (CS), Guiyang(GY), Fuzhou(FZ), Kunming(KM), Xiamen(XM), Guangzhou(GZ), Nanning(NN), Hainan(HN). (2) The genetic distances among populations relate to the collection sites to a great extent, though there are some exceptions. According to the facts that we know, (1) D. simulans is an exotic species; (2) The invasion of D. simulans in the mainland of China took place in recent 30 years or so. (3) The widespread distribution of D. simulans in the mainland of China started in recent 10 years or so. Therefore, the results above might not be due to geographical differentiation, but due to genetic differences of initial founder individual(s) between geographical populations. Some of geographically adjacent populations, however, have common initial founder individual(s), which results in the classification of D. simulans populations according to their geographical relationship. We suggest that there are two possible reasons which result in the genetic differences of initial founder individual(s) between geographical populations, one is the D. simulans in the mainland of China had multiple origins, the other is founder effect and/or bottleneck effect that the D. simulans have experienced in the course of expansion.